The field of this invention is nucleic acid sequence detection. The invention employs probes comprising photoactive analogues that can be incorporated into synthetic oligonucleotides during automated DNA synthesis for use in crosslinking of complementary nucleic acid sequences.
The amount of information concerning the genomes of a large variety of species is increasing exponentially. The availability of known sequences creates an enormous market for the detection of particular sequences present as DNA or RNA, whereby one can detect the presence of genes, their transcription products, polymorphic sequences, or mutations, such as lesions, substitutions, deletions, translocations, and the like. By knowing sequences of interest, one can detect a wide variety of pathogens, particularly unicellular microorganisms and viral strains, and genetic diseases including the presence of genes imparting antibiotic resistance to the unicellular microorganisms, as illustrative of only a few of the available possibilities. In addition, there are needs within the extensive areas of genetic counseling, forensic medicine, research, regulatory inspection of genetically modified organisms, and the like, for nucleic acid sequence detection technology.
In many instances, the target nucleic acid sequence is only a very small proportion of total nucleic acid in the sample. Furthermore, there may be many situations where the target nucleic acid of interest and other sequences present have substantial homology. It is therefore important to develop methods for the detection of the target nucleic acid sequence that are both sensitive and accurate.
Furthermore, oligonucleotide probe-based assay methods are known to depend upon careful optimization of the wash stringency. If the wash conditions are too stringent, then probe/target hybrids will be denatured, resulting in a decrease in the amount of signal in the assay. If the wash conditions are not sufficiently stringent, then non-specifically bound probes or mismatched probe/target hybrids will remain in the assay medium, resulting in high levels of non-specific or background signal in the assay. Optimal conditions are necessarily different for each probe because hybridization is a sequence-dependent phenomenon and would also depend on the extent to which near-homologous sequences are present in the sample.
The use of crosslinkable probes in nucleic acid hybridization assays to crosslink to target sequences is demonstrated in U.S. Pat. No. 4,826,967 by K. Yabusaki et al.; compounds are based on furocoumarin, (or psoralen) attached to existing polynucleotides (usually through adduct formation) and are satisfactory for many applications. However the crosslinking group/nucleoside adduct is difficult to synthesize particularly in large quantities. In U.S. Pat. No. 5,082,934, Saba et al. describe a photoactivatible nucleoside analogue comprising a coumarin moiety linked through its phenyl ring to the 1-position of a ribose or deoxyribose sugar moiety in the absence of an intervening base moiety. The resulting nucleoside analogue is used as a photo-crosslinking group when inserted into a polynucleotide as a replacement for one or more of the complementary nucleoside bases present in a probe used in hybridization assays. Nevertheless, new types of compounds that offer additional advantages, such as stability throughout probe synthesis and use, and conformational flexibility, continue to remain desirable.
There is, therefore, substantial interest in identifying alternative techniques which allow for the detection of specific DNA sequences and avoid the deficiencies of the other systems.
This invention provides non-nucleosidic, stable, photoactive compounds that can be used as photo-crosslinking reagents in nucleic acid hybridization assays, which may include genotyping and mutation detection assays, as well as techniques and intermediates that can be used to prepare the final products.
The compounds comprise coumarinyl derivatives prepared by linking the phenyl ring of a coumarin molecule or derivative to a hydroxy or polyhydroxy hydrocarbon molecule, such as one of the terminal hydroxy groups of a glycerol molecule. The (poly)hydroxy hydrocarbon moiety of the resulting compound is equivalent to the sugar of a nucleoside, while the coumarin moiety occupies the position of a base. It is to be understood that the (poly)hydroxy hydrocarbon moiety of the resulting compound is other than ribose or deoxy ribose. Accordingly, the compounds can be inserted into growing polynucleotide chains using automated (or manual) techniques of polynucleotide synthesis. The double bond between the 3 and 4 positions of the coumarin ring system is a photoactive group that covalently crosslinks to nucleosides in the complementary strand when an oligonucleotide containing this non-nucleoside analogue (the xe2x80x9ccrosslinkable probexe2x80x9d) is used in a hybridization assay and/or therapeutic application.
For the most part, the photoactive compound has the formula 
in which the substituents and linking groups are described below in more detail.
The (poly)hydroxy hydrocarbon backbones give maximum flexibility and stability to the oligonucleotide structure in which they are located as well as good solubility in aqueous and organic media.
Also provided herein are methods of use of the crosslinkable probe described herein. Methods and compositions are provided for detecting nucleic acid sequences by using the crosslinkable probe provided herein which comprises a cross linking system. Upon hybridization of the crosslinkable probe to the target and activation of the cross linking system, the crosslinkable probe and target are joined together by a covalent linkage. The method employs adding the crosslinkable probe to the target nucleic acid under conditions of base pairing and activating the crosslinking system, so that a covalent bond is formed between the target and the crosslinkable probe.
In a preferred embodiment, the crosslinkable probe includes a molecule that can be captured on a solid support, e.g., biotin, and thus can be captured on e.g., streptavidin-coated magnetic beads (a xe2x80x9ccapture probexe2x80x9d). In another preferred embodiment, one or more crosslinkable probes which also are complementary to the target nucleic acid sequence, include a reporter molecule, e.g., a fluorophore, an antigen, or other binding pair partner useful for direct or indirect detection methods (a xe2x80x9creporter probexe2x80x9d).